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Docetaxel inhibits SMMC-7721 human hepatocellular carcinoma cells growth and induces apoptosis
Chang-Xin Geng, Zhao-Chong Zeng, Ji-Yao Wang
Chang-Xin Geng, Ji-Yao Wang,
Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai
200032, China
Zhao-Chong Zeng,
Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai
200032, China
Correspondence to: Prof.
Ji-Yao Wang, Director of Department of Gastroenterology, Zhongshan Hospital,
Fudan University, Shanghai 200032, China. jiyao_wang@hotmail.com
Telephone: +86-21-64041990-2420
Fax: +86-21-34160980
Received:
2002-08-06 Accepted: 2002-09-03
Abstract
AIM: To investigate the in vitro anti-hepatocellular
carcinoma (HCC) activity of docetaxel against SMMC-7721 HCC cells and its
possible mechanism.
METHODS: The
HCC cells were given different concentrations of docetaxel and their growth was
measured by colony forming assay. Cell cycle and apoptosis were analyzed by flow
cytometry and fluorescence microscopy (acridine orange/ethidium bromide double
staining, AO/EB), as well as electronic microscopy. The SMMC-7721 HCC cell
reactive oxygen species (ROS) and glutathione (GSH) were measured after given
docetaxel.
RESULTS: Docetaxel
inhibited the hepatocellular carcinoma cells growth in a concentration dependent
manner with IC505≥10-10
M. Marked cell apoptosis and G2/M phase arrest were observed after treatment
with docetaxel ≥10-8M. Docetaxel promoted SMMC-7721 HCC cells ROS
generation and GSH deletion.
CONCLUSION: Docetaxel
suppressed the growth of SMMC-7721 HCC cells in vitro by causing apoptosis and
G2/M phase arrest of the human hepatoma cells, and ROS and GSH may play a key
role in the inhibition of growth and induction of apoptosis.
Geng CX, Zeng ZC, Wang JY. Docetaxel
inhibits SMMC-7721 human hepatocellular carcinoma cells growth and induces
apoptosis. World J Gastroenterol 2003; 9(4): 696-700
http://www.wjgnet.com/1007-9327/9/696.htm
INTRODUCTION
Decetaxel is a new taxoid structurally similar to
paclitaxel, a semisynthetic product of a renewable resource, the needles of the
European yew, Taxus baccata L. In comparison to paclitaxel, docetaxel is more
potent as an inhibitor of microtubule depolymerization[1]. Docetaxel
has shown an active effect against cancers[2-5]. Current studies have
shown that docetaxel combined with other chemotherapeutic drugs has higher
anticancer efficacy and reduced side effects in patients with breast,
pancreatic, gastric, urothelial carcinomas[6-9]. But there is limited
data about that in hepatocellular carcinoma and thus needs further
investigation. Human hepatocellular carcinoma (HCC) is one of the major causes
of death worldwide[10-12], and surgical resection is the only
curative therapy[13]. However it has limitations for patients with
multiple type or metastatic tumors. Searching for effective chemotherapeutic
agents is important to improve the survival rate of patients with advanced or
recurrent HCC after surgical treatment. HCC is usually insensitive to
chemotherapeutic drugs currently used in clinical setting, and there is thus an
urgent need for the evaluation of new active drugs against HCC. HCC is well
known for its expression of multidrug resitance (MDR) gene and its poor response
to chemotherapeutic agents, but it has been found that docetaxel is effective in
treating mice bearing xenografts of MDR protein positive human tumors[14],
also a low efflux of docetaxel from tumor cells being observed[15].
These results suggest that docetaxel may be effective in the treatment of HCC.
But the related information is limited and the mechanism of docetaxel remains to
be elucidated. This study is to investigate its growth inhibition and induction
of apoptosis effect and the mechanism in human SMMC-7721 hepatocellular cell
line, and to provide the theoretical basis for clinical application in patients
with HCC.
MATERIALS AND METHODS
Reagents
Docetaxel (Rhone-Poulenc Rorer
Pharmaceuticals Inc.) was stored as 100 mm
stock solution in absolute ethanol at -80 ℃.
This solution was further diluted in the medium and used in the cell culture
immediately before each experiment.
Cell cultures
Human hepatocellular carcinoma SMMC-7721 cell
line was obtained from Liver Cancer Institute of Fudan University. The cells
were cultured in flasks with RPMI1640 (Gibco BRL, Grand Island, NY) medium
supplemented with 100 IU/ml penicillin, 100 mg/ml
streptomycin and 10 % new born bovine serum (Hyclone, Logan, UT) and maintained
at 37 ℃
in humidified atmosphere containing 5 % CO2.
Colony forming assay
Exponential growth phase SMMC-7721 cells were
seeded in Ф60 mm culture plate (Corning, NY) at a density of 200 cells/plate,
after 24hr, the media was discarded and replaced with equal volume of fresh
media containing different doses of docetaxel (0, 10-11, 10-10,
10-9, 10-8, 10-7, 10-6M). 24hr later
docetaxel containing media was again discarded and replaced by media without
containing docetaxel. The cells were cultured continually for 9 days. The
colonies were fixed with 95 % ethanol and stained with Giemsa, and manually
counted. Colonies ≥50 cells were considered survivors. All experiments were
conducted in triplicate.
Flow cytometric analysis of DNA
content after docetaxel treatment
After exponential growth phase SMMC-7721
cells were treated with different doses of docetaxel (0, 10-11, 10-10,
10-9, 10-8, 10-7, 10-6M) for 24hr
and harvested with 0.25 % trypsin and resuspended in RPMI1640 media. 1×106 cells was centrifuged at
300 g for 5 minutes and then washed once with PBS .The cell pellets were added
with 100 % precooled ethanol at 4 ℃
for 4hr, centrifuged at 300 g for 5 minutes and then washed once, resuspended
with PBS (1×106cells/ml). The cells were
added 500 u Rnase, incubated at 37 ℃
for 30 minutes, washed with PBS. 1?06cells were stained with 50 mg/ml
PI for 20 minutes in the dark. The DNA content of each cell was measured using a
Becton Dickinson FACSCalibur flow cytometer and analyzed with ModFit LT software
(San Jose, CA).
Morphological study with fluorescence
microscope
Exponential growth phase SMMC-7721 cells were
treated with 10nM docetaxel for 24hr and 48hr and harvested with 0.25 % trypsin
and resuspended in RPMI1640 medium. 1?06 cells were washed once and resuspended
with PBS. 25 ml of
the cells suspension was mixed with 1 ml
of dye mixture containing AO 100 mg/ml
and EB 100 mg/ml
in PBS[16]. The cells were visualized immediately under a
fluorescence microscope, the peak excitation wave length was 490 nm.
Transmission electron microscopic
observation
After exponential growth phase SMMC-7721
cells treated with 10nM docetaxel for 24hr and 48hr, they were fixed in
Karnovsky solution, followed by cacodylate buffer for ultrastructural
examination. The cells were postfixed in 1 % osmium tetroxide and dehydrated for
staining with uranyl acetate and lead citrate. Thin sections were observed under
electron microscope.
ROS measurement
After exponential growth phase SMMC-7721
cells were treated with different doses of docetaxel (0.25 nM, 0.5 nM, 10 nM)
for 24hr, the cells were incubated with 2? 7?dichlorofluorescin diacetate (DCF/DA;
5 mm;
Sigma) at 37 ℃
for 50 minutes to estimate the ROS level. The cells were harvested and detected
immediately for fluorescence intensity detection on Becton Dickinson FACSCalibur
flow cytometer and data were acquired and analyzed using FACS/CELLQuest software
(San Jose, CA) on a Power Macintosh 7600/120 computer (Apple Computers,
Cupertino, CA)[17], three independent experiments were repeated in
each drug treated group and the control group was not given docetaxel but
incubated with DCF/DA. All experiments were conducted in triplicate.
Determination of intracellular
GSH content
The determination of intracellular GSH
content was conducted according to Shen et al[18] with
modification. Briefly after logarithmic growth phase SMMC-7721 cells (≥106)
were treated with different doses of docetaxel (0.25 nM, 0.5 nM, 10 nM) for
24hr, harvested by trypsination, washed, resuspended in PBS and counted under
phase contrast microscope. The cells suspension was centrifuged at 300 g for 5
minutes, the cell pellets were added 0.75 ml distilled water and 0.25 ml
thiosalicylic acid to precipitate the protein. After centrifugation at 12 000 g
for 5 minutes at 4 ℃,
the supernatant was used for GSH assay. The control group was not given
docetaxel. All experiments were conducted in triplicate.
Statistical analysis
The intracellular GSH content was expressed
as mean ±SD. Their
differences between drug treated groups and the control group were analyzed with
Student's t-test.
RESULTS
Effect of docetaxel on SMMC-7721 cells
growth inhibition
The colony forming ability decreased
gradually with increasing dose of docetaxel treatment, when the docetaxel
concentration was increased to 10-8M, the colony forming inhibition
rate attained 100 % with IC50 5×10-10 M (0.5 nM) (Figure 1).
Figure
1 (PDF) The survival fraction of SMMC-7721
cells treated with docetaxel for 24hr.
Effect on of docetaxel cell cycle and
apoptosis of SMMC-7721 cells
As demonstrated in Figure 2, docetaxel
induced a marked apoptosis at 10-8M for 24hr, and caused cells G2/M phase arrest
mainly at 10-7M and 10-6M, at 10-6M the G2/M
phase cells accounted for 96.1 %.
Morphological observation with
fluorescence microscope
After the SMMC-7721 cells were given 10-8M
docetaxel for 24hr, the early apoptotic cells could be observed: because of
their cells membrane integrity, the cells were stained green with AO, the nuclei
exhibited bright condensed chromatin or fragmented, some cells blebbed. After
the cells being treated for 48hr, some late apoptotic cells could be observed:
Since their cell membrane lost integrity, they were stained red with EB, the
apoptotic bodies could be seen clearly. On the contrary, the untreated cells did
not show these apoptotic characteristics(Figure 3).
Figure
2 (PDF) Cell cycle changes and apoptosis induction in SMMC-7721 cells
treated with docetaxel for 24hr. Cells were treated with docetaxel at 0M(A), 10-11
M(B), 10-10 M(C), 10-9 M(D), 10-8 M(E), 10-7
M(F), 10-6M(G). A marked apoptosis was induced at 10-8 M,
and a peaked G2/M phase accumulation was caused at 10-6M.
Ap:apoptosis.
Figure 3 Morphological
study with fluorescence microscope. After the SMMC-7721 cells were given 10-8M
docetaxel for 24hr(A), the
cells were stained green with AO, the nuclei exhibited bright condensed
chromatin or fragmented, some cells blebbed. After being treated for 48hr (B),
cells were stained red with EB, the apoptotic bodies could be seen clearly. On
the contrary, the untreated cells (C)
did not show these apoptotic characteristics.
Transmission electron microscopic
observation
After treatment with 10-8M
docetaxel for 24hr, the chromatin of some SMMC-7721 cells was located along the
nuclear edges or formed irregularly shaped crescents at the nuclear edges, or
became condensed or fragmented; Some cells nuclear membrane became irregular;
The vacuole in some cytoplasma could be observed (Figure 4).
Effect of docetaxel on intracellular
ROS
As demonstrated in Figure 5, The SMMC-7721
cells ROS level(corresponding to the fluorescence intensity) increased after
treatment with 0.25 nM, 0.5 nM, 10 nM docetaxel. In comparison with the control
group, the ROS level increased by 1.69, 1.78 and 1.80 times respectively.
Figure 4 Transmission
electron microscopic observation. After treatment with 10-8M
docetaxel for 24hr, the chromatin of some SMMC-7721 cells was located along the
nuclear edges or formed irregularly shaped crescents at the nuclear edges (A,C),or
became condensed or fragmented (A, B); The vacuole could be observed in some
cytoplasm (A, C).
Figure
5 (PDF) The
effect of docetaxel on the formation of ROS in SMMC-7721 cells. Data are from
three independent experiments.
Effect of docetaxel on intracellular
GSH
The control group intracellular GSH content
was 11.6±1.1 fmol/cell, after treatment with
docetaxel 0.25 nM, 0.5 nM, 10 nM for 24hr, the SMMC-7721 cells GSH decreased to
4.7±0.7 fmol/cell, 3.0±0.5 fmol/cell, 1.0±0.1 fmol/cell respectively, there was a
significant difference between the drug treated groups and the control group (P<0.05).
DISCUSSION
In the present study, we used colony forming
assay to observe the effect of docetaxel on the growth of the human
hepatocellular carcinoma SMMC-7721 cells in vitro, indicating that doctaxel can
inhibit the SMMC-7721 cells growth and in concentration dependent manners, at 10-8M the survival fraction decreased to 0 (Figure1). A previous study
demonstrated that docetaxel showed cytotoxic effect on other human hepatoma cell
lines[17], together with this report, our result suggests that the
growth-inhibiting effect of docetaxel may be a general phenomenon for human
hepatocellular carcinoma cells. Also the flow cytometry showed that docetaxel
causes SMMC-7721 cells apoptosis at a low dose (10-8M), which was confirmed by
fluorescence microscope, transmission electron microscope, and at 10-7M, 10-6M
induced a marked G2/M phase arrest without apparent apoptosis, this phenomenon
seems to suggest that docetaxel can induce apoptosis independent of G2/M phase
arrest in SMMC-7721 cells.
In order to elucidate the mechanisms of
the anti-hepatocellular carcinoma action of docetaxel, we examined its possible
effect on cell cycle kinetics of SMMC-7721 cells. As demonstrated in Figure 2,
following treatment with docetaxel for 24hr, the G2/M phase cells increased with
docetaxel doses increasing from 10-8M, at 10-6M the G2/M phase cells accounted
for 96.1 %. This result indicates that high dose of docetaxel blocks cell cycle
at G2/M phase, disturbs mitosis and inhibits SMMC-7721 cells growth. In this
clinical setting, such information may be useful for liver carcinoma therapy
with docetaxel and other cytotoxic drugs which affect cell cycle progression.
According to Figure 1, docetaxel could
inhibit SMMC-7721 cells at very low doses, the IC50 is 5×10-10M. This dose of
docetaxel had no significant effect on the cell apoptosis or cell cycle
progression, which was confirmed by flow cytometry. In order to elucidate the
mechanism underlying this phenomenon, the intracellular ROS and GSH were further
investigated in this study. Our results indicated that docetaxel both at 1/2IC50,
IC50 and apoptosis-inducing dose (10nM) could cause significantly
increased ROS levels (Figure 5) and significantly decreased GSH, compared with
those of the control.
It has been reported that tumor cells
have persistently higher levels of ROS[19]. The relationship between ROS and
apoptosis, cell growth inhibition has been broadly investigated. ROS has been
found to regulate translocation of NF-kB[20] as well as translocation of p53 and
the p53-mediated apoptosis pathway[21,22]. Also it has been found that induction
of apoptosis was accompanied with the generation of intracellular ROS prior to
the activation of caspase-3[23] and down-regulation of Bcl-2[24]. Simizu et
al. reported that some anticancer agents, including vinblastin and
camptothecin, induced cells apoptosis with the generation of ROS[25]. Recently
it was reported that arsenic trioxide and l-S,R-buthionine sulfoximine (BSO)combination
treatment enhanced hepatocellular carcinoma cells apoptosis through increased
production of ROS[26], Also it was reported ROS played a mediatory role in the
synergistic interaction between 1, 25-dihydroxyvitamin D (3) and anticancer
cytokines[27], modulation of the ROS production intra and extracellularlly may
influence the cell survival during oxidative insults[28].
Accordingly, we speculate that docetaxel-induced
high level ROS may be involved in the growth inhibition and apoptosis of
SMMC-7721 cells.
In order to confirm the SMMC-7721 cells
redox status after docetaxel treatment, the intracellular GSH content was also
investigated. Our result demonstrated that the intracellular GSH decreased
significantly (P<0.05) after both 1/2IC50, IC50 and apoptosis-inducing
dose docetaxel treatment, compared with that of the control group. This
indicates that the decrease of GSH may be involved in the SMMC-7721 cells growth
inhibition and apoptosis caused by docetaxel. Liu et al. found Salvia
miltiorrhiza inhibited human hepatoma HepG2 cells growth and induced apoptosis
involving in intracellular GSH deletion[29], early studies have also
demonstrated that the onset of apoptosis was associated with a fall of
intracellular GSH in different cellular systems[30]. And GSH could significantly
reduce apoptosis mediated by a monoclonal antibody directed to Fas antigen, and
apoptosis could be nearly completely prevented by GSH[31]. GSH is an important
cellular thiol which is regarded as the major determinant of the intracellular
redox potential, on the other hand, apoptosis may be regulated by the reodx
status within the cells[32]. Loss of GSH was shown to be tightly coupled with a
number of down-stream events in apoptosis, in caspase activation and events in
chromatin[33], and GSH depletion may act as a link between oxidative stress and
ceramide-mediated apoptosis[34]. Also it was found that decreased cells
proliferation associated with decreased levels of intracellular GSH, and
blockade of GSH synthesis enhanced ROS production and suppressed cells
proliferation[35], cells with compromised cellular GSH were susceptible to redox
imbalance-induced inhibition of proliferation[36].
In summary, the present study
demonstrates that docetaxel has profound effects on SMMC-7721 hepatocellular
carcinoma cells in vitro. It reduces the proliferation of these cells, causes
changes in their morphology, and induces cell death by apoptosis. It leads to
ROS generation and GSH deletion, subsequently results in redox imbalance in
SMMC-7721 cells, and it is speculated that the redox imbalance caused by
docetaxel may play an important role in the growth inhibition and induction of
apoptosis of SMMC-7721 cells. As far as we know, there is few report concerning
the redox imbalance and docetaxel. Our results not only provide the basis for
further in vivo and clinical research in hepatocellular carcinoma, but also
contribute to understanding of the pharmacology of docetaxel further.
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Edited by Wu XN
